Ongoing study on the chemical constituents of the roots of Macleaya microcarpa led to the isolation of eight compounds of derivatives of triterpenes and organic acids in addition to some previously identified benzophenanthridines. The eight compounds were identified by spectroscopic methods as well as comparison with literature values as 1-oxo-2, 22 (30)-hopandien-29-oic acid (1), 3-oxo-12-oleanen-30-oic acid (2), 3α-hydroxy-12-oleanen-30-oic acid (3), 3β-hydroxy-12-oleanen-30-oic acid (4), ferulic acid (5), ferulic acid 4-O-β-D-glucoside (6), 3-O-feruloylquinic acid (7), and methyl 3-O-feruloylquinate (8). Of which, 1 is a new triterpenoid of hopanes and 2-8 are isolated from M microcarpa for the first time. In order to discover natural active compounds as potential agents of anti-ulcerative colitis (UC), an in vitro drug high-throughput screening model targeted x-box-binding protein 1 (xbp1) was employed to evaluate the activity of the major chemical constituents of M microcarpa. The result confirmed that two dihydrobenzophenanthridines, dihydrosanguinarine (9) and dihydrochelerythrine (10), showed a certain activity on activating the transcription of xbpl, a transcription factor (TF) associated with the occurrence, development, and potential treatment of UC, with their relative activating ratios being 1.76 and 1.77 times, respectively, as compared with control group.
Anti-Ulcer Agents ; chemistry ; Benzophenanthridines ; chemistry ; DNA-Binding Proteins ; genetics ; Isoquinolines ; chemistry ; Papaveraceae ; chemistry ; Plant Roots ; chemistry ; Regulatory Factor X Transcription Factors ; Transcription Factors ; genetics ; Transcription, Genetic ; Triterpenes ; chemistry

OBJECTIVETo study the molecular mechanism of differentiation induction by 2-methoxyestradiol (2ME2) of myeloma cell lines.

METHODSDifferentiation induction effect on myeloma cell lines LP-1, CZ-1 and NCI-H929 which were incubated with 2ME2 and XBP-1, Blimp-1, pax-5 phosphorothioate antisense oligodeoxynucleotide (ASODN) was evaluated by cell morphology, CD49e expression, quantitation of light chain secretion, and the level of pax-5 and XBP-1 mRNA expression.

RESULTS2ME2 caused morphological, immunophenotypic and the supernatant light chain secretion changes typical of differentiation in all the three myeloma cell lines. 2ME2 up-regulated the XBP-1 mRNA expression. XBP-1 and Blimp-1 ASODNs partially inhibited the differentiation of LP-1, CZ-1, NCI-H929 cells induced by 2ME2; whereas pax-5 ASODN did the contrary. After incubated with pax-5 ASODN for 72 hours, LP-1, CZ-1, NCI-H929 cells exhibited characteristic morphologic feature of differentiation. The expression of CD49e was increased statistically (P < 0.05). Light chain secretion in the supernatant was also increased statistically (P < 0.05). After incubation with Blimp-1 ASODN, the level of XBP-1 mRNA was declined, while the level of pax-5 mRNA increased.

CONCLUSION2ME2 could induce cell differentiation and up-regulate XBP-1 mRNA expression in myeloma cell lines. Blimp-1 could help 2ME2 with inducing differentiation of myeloma cells through downregulating pax-5 mRNA and upregulating XBP-1 mRNA.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; metabolism ; Estradiol ; pharmacology ; Humans ; Multiple Myeloma ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; Regulatory Factor X Transcription Factors ; Transcription Factors ; genetics ; metabolism ; X-Box Binding Protein 1

The aim of this study was to explore the effect of 2 different spliceosomes of X-box binding protein 1 (XBP-1), the spliced form XBP-1s and unspliced form XBP-1u, on myeloma cell differentiation and its mechanism. The overexpression plasmids pcDNA3.1-C-XBP1u and pcDNA3.1-C-XBP1s were constructed and transfected into myeloma cell line U266, RPMI8226. The morphology of U266 and RPMI 8226 cells was observed by means of light microscope, the expression rate of CD49e on cell surface was detected by flow cytometry, the ELISA was used to determine the changes of light chain protein level in supernatants of cell culture, the Western blot was used to assay the expression changes of XBP1u and XBP1s. The results showed that the overexpression of XBP1u could promote the myeloma cell differentiation morphologically displaying the maturation of plasmocytes, the CD49e positive expression rates on surface of U266 and RPMI8226 cells were obviously up-regulated from 9.02±0.3% and 5.17±0.92% in control group to 27.7±1.14% and 13.97±1.79% respectively (p<0.01), the levels of light chain protein in supernatants of U266 and RPMI 8226 cell cultures increased from 474.75±19.52 ng/ml and 289.44±6.19 ng/ml in control group to 692.34±21.17 ng/ml and 401.55±13.7 ng/ml respectively (p<0.01, p<0.05), while the above-mentioned parameters in the overexpressed XBP-1s showed no significant changes, which indicated no promotive effect of overexpressed XBP1s on myeloma cell differentiation. It is concluded that the up-regulation of XBP-1u expression plays an important role in the differentiation of myeloma cells.
Cell Differentiation ; genetics ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Multiple Myeloma ; genetics ; pathology ; Regulatory Factor X Transcription Factors ; Spliceosomes ; genetics ; Transcription Factors ; genetics ; Transfection ; X-Box Binding Protein 1

This study was purposed to investigate the effect of xbp-1 gene silencing on bortezomib-induced apoptosis in multiple myeloma cell line NCI-H929 (H929). After xbp-1 gene expression was interfered by small hairpin RNA, the cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining, and the expression level of XBP-1 protein was detected by Western blot. The results showed that XBP-1 protein level of H929 cells was inhibited effectively by the PLL3.7 lentiviral vector mediated expression xbp-1 shRNA. The apoptosis rate was significantly higher in xbp-1 shRNA-expressing cells than in untreated control group [(10.13±0.61)% vs (2.5±0.2)%, p<0.05]. After treatment with bortezomib, the apoptosis rate of XBP-1 protein functionally deficient H929 cells was significantly higher than those in vector control group [(45.07±1)% vs (19.53±0.8)%, p<0.05]. It is concluded that xbp-1 gene silencing can significantly enhance the pro-apoptotic activity of bortezomib in multiple myeloma cells.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; Gene Silencing ; Humans ; Multiple Myeloma ; genetics ; Pyrazines ; pharmacology ; RNA, Small Interfering ; genetics ; Regulatory Factor X Transcription Factors ; Transcription Factors ; genetics ; X-Box Binding Protein 1

The saturated free fatty acid (FFA), palmitate, could induce apoptosis in various cell types, but little is known about its effects on human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). Here, we investigated whether palmitate induced apoptosis and endoplasmic reticulum (ER) stress in hUC-MSCs. hUC-MSCs were stained by labeled antibodies and identified by flow cytometry. After administration with palmitate, apoptotic cell was assessed by flow cytometry using the Annexin V-FITC/7-AAD apoptosis detection kit. Relative spliced XBP1 levels were analyzed using semi-quantitative RT-PCR. The mRNA of BiP, GRP94, ATF4 and CHOP were analyzed by real-time PCR. Relative BiP and CHOP protein were analyzed using Western blot analysis. The results showed that hUC-MSCs were homogeneously positive for MSC markers; palmitate increased apoptosis of hUC-MSCs and activated XBP1 splicing, BiP, GRP94, ATF4 and CHOP transcription. These findings suggest that palmitate induces apoptosis and ER stress in hUC-MSCs.
Activating Transcription Factor 4 ; metabolism ; Apoptosis ; DNA-Binding Proteins ; metabolism ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; metabolism ; Humans ; Membrane Glycoproteins ; metabolism ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Palmitates ; pharmacology ; Regulatory Factor X Transcription Factors ; Transcription Factor CHOP ; metabolism ; Transcription Factors ; metabolism ; Umbilical Cord ; cytology ; X-Box Binding Protein 1

BACKGROUNDPrediabetes is an early stage of β-cell dysfunction presenting as insulin resistance. Evidences suggest that endoplasmic reticulum (ER) stress is involved in the pathogenesis of type 2 diabetes mellitus and prediabetes. In a Chinese population with prediabetes, we investigated single nucleotide polymorphisms (SNPs) in the genes of PERK, JNK, XBP1, BIP and CHOP which encode molecular proteins involved in ER stress pathways.

METHODSNine SNPs at the PERK, JNK, XBP1, BIP and CHOP loci were genotyped by mass spectrometry in 1 448 unrelated individuals. By using a 75 g oral glucose tolerance test (OGTT), 828 subjects were diagnosed as prediabetes and 620 subjects aged 55 years and over as normal controls based on WHO diagnostic criteria (1999) for diabetes mellitus.

RESULTSThe allele C of SNP rs867529 at PERK locus was a risk factor for prediabetes, with the carriers of C allele genotype at a higher risk of prediabetes compared to non-carriers (OR = 1.279, 95% CI: 1.013-1.614, P = 0.039, after adjustment for age, sex and body mass index (BMI). The SNPs rs6750998 at PERK locus was associated with homeostasis model assessments of insulin resistance (HOMA-IR) (P = 0.019), and rs17037621 with BMI (P = 0.044). The allele G of SNP rs10986663 in BIP gene was associated with a decreased risk of prediabetes (OR = 0.699, 95% CI: 0.539-0.907, P = 0.007). The SNP rs2076431 in JNK gene was associated with fasting plasma glucose levels (P = 0.006) and waist-hip ratios (P = 0.019). The SNP rs2239815 in XBP1 gene was associated with 2-hour plasma glucose levels after 75 g oral glucose load (P = 0.048) in the observed population.

CONCLUSIONCommon variants at PERK and BIP loci contributed to the risk of prediabetes, and the genetic variations in JNK and XBP1 genes are associated with diabetes-related clinical parameters in this Chinese population.

Aged ; DNA-Binding Proteins ; genetics ; Diabetes Mellitus, Type 2 ; genetics ; Female ; Genotype ; Humans ; MAP Kinase Kinase 4 ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Prediabetic State ; genetics ; Regulatory Factor X Transcription Factors ; Transcription Factor CHOP ; genetics ; Transcription Factors ; genetics ; X-Box Binding Protein 1 ; eIF-2 Kinase ; genetics

OBJECTIVETo analyze the transcription activation and possible regulation mechanism of human X-box binding protein 1(XBP1)gene 5'upstream DNA sequence in different cell lines.

METHODSSix kinds of XBP1 promoter deletion mutants were cloned into pGEM-Teasy vector, which included XBP1 gene 5' upstream -1039 to 66 bp,-859 to 66 bp,-623 to 66 bp,-351 to 66 bp,-227 to 66 bp,-227 to -45 bp respectively. Every deletion mutant sequence was cut from Teasy-XBP1p by KpnI and Xho I, and subcloned into pCAT3-Basic to produce a set of constructs termed as p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p, respectively. The transcription activity of each construct was detected after transiently transfecting K562, HepG2,NIH-3T3 and L0(2)cell with FuGENE 6 transfection reagent. Cells transfected by pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of chloramphenicol acetyltransferase(CAT), which reflects the transcription activation of the XBP1 gene promoter, was detected by ELISA after 48 hours of transfection.

RESULTSThe reporter vectors of six kinds of XBP1 promoter deletion mutants were successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activities of p4-XBP1p and p5-XBP1p were higher than the other deletion mutants in K562 and HepG2. And the activity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3.

CONCLUSIONThe XBP1 gene promoter can transactivate its downstream gene to transcription. The core sequence of XBP1 promoter was implied between -227 bp and 66 bp. This sequence was connected with the transcriptional activity of XBP1 promoter closely. Its transcription activity varies with different cell lines. XBP1 promoter might drive gene expression with cell-type specificity.

3T3 Cells ; 5' Flanking Region ; genetics ; Animals ; Base Sequence ; Cell Line ; Chloramphenicol O-Acetyltransferase ; metabolism ; DNA ; analysis ; DNA-Binding Proteins ; genetics ; Gene Deletion ; Gene Expression Regulation ; physiology ; Genes, Reporter ; Humans ; K562 Cells ; Mice ; Molecular Sequence Data ; Nuclear Proteins ; genetics ; Promoter Regions, Genetic ; genetics ; Regulatory Factor X Transcription Factors ; Transcription Factors ; Transcription, Genetic ; physiology ; Transcriptional Activation ; Transfection ; Tumor Cells, Cultured ; X-Box Binding Protein 1

The purpose of the present study is to explore the effect of oxidized low density lipoprotein (ox-LDL) on the induction of endoplasmic reticulum stress (ERS) and the underlying mechanisms in ox-LDL-induced macrophage foam-forming process. RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum, and then treated with ox-LDL (25, 50 and 100 mg/L), anti-CD36 monoclonal antibody+ox-LDL and tunicamycin (TM), respectively. After incubation for 24 h, the cells were collected. The cellular lipid accumulation was showed by oil red O staining and the content of cellular total cholesterol was quantified by enzymatic colorimetry. The expression of glucose-regulated protein 94 (GRP94), a molecular marker of ERS, was determined by immunocytochemistry assay. The levels of GRP94 protein, phosphorylated inositol-requiring enzyme 1 (p-IRE1) and X box binding protein 1 (XBP1) in RAW264.7 cells were detected by Western blotting. The results indicated that after incubation with ox-LDL (25, 50 and 100 mg/L) for 24 h, a large amount of lipid droplets were found in the cytoplasm, and the contents of cellular total cholesterol were increased by 2.1, 2.8 and 3.1 folds compared with the control, respectively. Anti-CD36 antibody decreased markedly the cellular lipid accumulation induced by ox-LDL at 100 mg/L. Both ox-LDL and TM, a specific ERS inducer, could up-regulate the protein expression of GRP94 in a dose-dependent manner. Furthermore, p-IRE1 and XBP1, two key components of the unfolded protein response, were also significantly induced by the treatment with ox-LDL. The up-regulations of the three proteins induced by ox-LDL were inhibited significantly when the macrophages were pre-incubated with anti-CD36 antibody. These results suggest that ox-LDL may induce ERS in a dose-dependent way and subsequently activate the unfolded protein response signaling pathway in RAW264.7 macrophages, which is potentially mediated by scavenger receptor CD36.
Animals ; CD36 Antigens ; physiology ; Cell Line ; Cells, Cultured ; DNA-Binding Proteins ; metabolism ; Endoplasmic Reticulum ; drug effects ; Foam Cells ; cytology ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Protein-Serine-Threonine Kinases ; metabolism ; Regulatory Factor X Transcription Factors ; Stress, Physiological ; drug effects ; Transcription Factors ; metabolism ; X-Box Binding Protein 1

Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.
Breast Neoplasms ; genetics ; metabolism ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; metabolism ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Humans ; Protein Interaction Domains and Motifs ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Regulatory Factor X Transcription Factors ; Signal Transduction ; Transcription Factors ; genetics ; metabolism ; X-Box Binding Protein 1

OBJECTIVETo explore the role of Bid protein in the mitochondria and endoplasmic reticulum (ER) associated apoptotic pathway.

METHODSApoptosis of MUTZ-1 cells induced by homoharringtonine (HHT) was measured by FACS. Mitochondria and ER associated apoptotic pathway was detected by RT-PCR and Western blotting. And the translocation of Bid protein was measured by laser scanning confocal microscope (LSCM).

RESULTSAfter exposure of MUTZ-1 to HHT at 0.05 microg/ml for 24 h, typical ER-stress phenomenon induced apoptotic cells and release of Ca2+ from the cytosolic Ca2+ storage and the loss of mitochondrial membrane potential were observed. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor were markedly increased at 4 h after 0.05 microg/ml HHT treatment and peaked at 12 h, then decreased steady. Activation of caspase protein was also observed at 8 h. The translocation of Bid protein from ER to mitochondria was observed at 12 h after HHT treatment.

CONCLUSIONHHT can induce MUTZ-1 cells apoptosis. The cell death may be likely mediated by the ER stress pathway as well as mitochondrial pathway and Bid protein may be the cross talk of the two apoptotic pathways.

Apoptosis ; drug effects ; physiology ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; physiology ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Caspases, Initiator ; metabolism ; Cell Line ; DNA-Binding Proteins ; metabolism ; Endoplasmic Reticulum ; metabolism ; physiology ; Harringtonines ; pharmacology ; Humans ; Mitochondria ; metabolism ; physiology ; Regulatory Factor X Transcription Factors ; Transcription Factors ; metabolism